Background: Recent studies using batch-fermentation suggest that the red macroalgae Asparagopsis taxiformis has the potential to reduce methane (CH 4) production from beef cattle by up to~99% when added to Rhodes grass hay; a common feed in the Australian beef industry. These experiments have shown significant reductions in CH 4 without compromising other fermentation parameters (i.e. volatile fatty acid production) with A. taxiformis organic matter (OM) inclusion rates of up to 5%. In the study presented here, A. taxiformis was evaluated for its ability to reduce methane production from dairy cattle fed a mixed ration widely utilized in California, the largest milk producing state in the US. Results: Fermentation in a semi-continuous in-vitro rumen system suggests that A. taxiformis can reduce methane production from enteric fermentation in dairy cattle by 95% when added at a 5% OM inclusion rate without any obvious negative impacts on volatile fatty acid production. High-throughput 16S ribosomal RNA (rRNA) gene amplicon sequencing showed that seaweed amendment effects rumen microbiome consistent with the Anna Karenina hypothesis, with increased β-diversity, over time scales of approximately 3 days. The relative abundance of methanogens in the fermentation vessels amended with A. taxiformis decreased significantly compared to control vessels, but this reduction in methanogen abundance was only significant when averaged over the course of the experiment. Alternatively, significant reductions of CH 4 in the A. taxiformis amended vessels was measured in the early stages of the experiment. This suggests that A. taxiformis has an immediate effect on the metabolic functionality of rumen methanogens whereas its impact on microbiome assemblage, specifically methanogen abundance, is delayed. Conclusions: The methane reducing effect of A. taxiformis during rumen fermentation makes this macroalgae a promising candidate as a biotic methane mitigation strategy for dairy cattle. But its effect in-vivo (i.e. in dairy cattle) remains to be investigated in animal trials. Furthermore, to obtain a holistic understanding of the biochemistry responsible for the significant reduction of methane, gene expression profiles of the rumen microbiome and the host animal are warranted.

With increasing interest in feed-based methane mitigation strategies and regional legal directives aimed at methane production from the agricultural sector, identifying local sources of biological feed additives will be critical for rendering these strategies affordable. In a recent study, the red alga Asparagopsis taxiformis harvested offshore Australia was identified as highly effective for reducing methane production from enteric fermentation. Due to potential difference in methane-reduction potential and the financial burden associated with transporting the harvested seaweed over long distances, we examined locally sourced red seaweed A. taxiformis and brown seaweed Zonaria farlowii for their ability to mitigate methane production when added to feed widely used in the Californian dairy industry. At a dose rate of 5% dry matter (DM), California-sourced A. taxiformis and Z. farlowii reduced methane production by up to 74% (p < 0.05) and 11% (p < 0.05) during in vitro rumen fermentation, respectively. No effect on CO 2 production was observed for either seaweed. The measured decrease in methane production induced by A. taxiformis and Z. farlowii amendment, suggest that these local macroalgae are indeed promising candidates for biotic methane mitigation strategies in California, the largest milk producing state in the United States. To determine their real potential as methane mitigating feed supplements in the dairy industry, their effect in vivo will need to be investigated.

Background: Recent studies using batch-fermentation suggest that the red macroalgae Asparagopsis taxiformis might reduce methane (CH 4 ) emission from beef cattle by up to ~99% when added to rhodes grass hay, a common feed in the Australian beef industry. These experiments have shown significant reductions in methane without compromising other fermentation parameters (i.e. volatile fatty acid production) with A. taxiformis organic matter (OM) inclusion rates of up to 5%. In the study presented here, A. taxiformis was evaluated for its ability to reduce methane production from dairy cattle fed a mixed ration widely utilized in California; the largest milk producer in the US.Results: Fermentation in a semi-continuous in-vitro rumen system suggests that A. taxiformis can reduce methane production from enteric fermentation in dairy cattle by 95% when added at a 5% OM inclusion rate without any obvious negative impacts on volatile fatty acid production. High-throughput 16S ribosomal RNA (rRNA) gene amplicon sequencing showed that seaweed amendment effects rumen microbiome communities consistent with the Anna Karenina hypothesis, with increased beta-diversity, over time scales of approximately three days. The relative abundance of methanogens in the fermentation vessels amended with A. taxiformis decreased significantly compared to control vessels, but this reduction in methanogen abundance was only significant when averaged over the course of the experiment. Alternatively, significant reductions of methane in the A. taxiformis amended vessels was measured in the early stages of the experiment. This suggests that A. taxiformis has an immediate effect on the metabolic functionality of rumen methanogens whereas its impact on microbiome assemblage, specifically methanogen abundance, is delayed. Conclusions:The methane reducing effect of A. taxiformis during rumen fermentation makes this macroalgae a promising candidate as a biotic methane mitigation strategy in the largest milk producing state in the US. But its effect in-vivo (i.e. in dairy cattle) remains to be investigated in animal trials. Furthermore, to obtain a holistic understanding of the biochemistry responsible for the significant reduction of methane, gene expression profiles of the rumen microbiome and the host animal are warranted.

There has been a great interest to identify a microbial marker that can be used to predict feed efficiency of beef cattle. Such a marker, specifically one that would allow an early identification of animals with high feed efficiency for future breeding efforts, would facilitate increasing the profitability of cattle operations and simultaneously render them more sustainable by reducing their methane footprint. The work presented here suggests that Prevotella copri might be an ideal microbial marker for identifying beef cattle with high feed efficiency early in their life span and in the production cycle. Developing more refined quantification techniques that allow correlation of P. copri to feed efficiency of beef cattle that can be applied by lay people in the field holds great promise to improve the economy of cattle operations while simultaneously reducing their environmental impact by mitigating methane production from enteric fermentation.

34With increasing interest in feed based methane mitigation strategies, fueled by local legal 35 directives aimed at methane production from the agricultural sector in California, identifying 36 local sources of biological feed additives will be critical in keeping the implementation of these 37 strategies affordable. In a recent study, the red alga Asparagopsis taxiformis stood out as the 38 most effective species of seaweed to reduce methane production from enteric fermentation. Due 39 to the potential differences in effectiveness based on the location from where A. taxiformis is 40 collected and the financial burden of collection and transport, we tested the potential of A. 41 taxiformis, as well as the brown seaweed Zonaria farlowii collected in the nearshore waters off 42 Santa Catalina Island, CA, USA, for their ability to mitigate methane production during in-vitro 43 rumen fermentation. At a dose rate of 5% dry matter (DM), A. taxiformis reduced methane 44 production by 74% (p ≤ 0.01) and Z. farlowii reduced methane production by 11% (p ≤ 0.04) 45 after 48 hours and 24 hours of in-vitro rumen fermentation respectively. The methane reducing 46 3effect of A. taxiformis and Z. farlowii described here make these local macroalgae promising 47 candidates for biotic methane mitigation strategies in the largest milk producing state in the US. 48To determine their real potential as methane mitigating feed supplements in the dairy industry, 49 their effect in-vivo requires investigation. 50 51